Agent for inhibiting production of hepatitis c virus and its use

ABSTRACT

This invention provides an agent for inhibiting production of hepatitis C virus with notable anti-HCV activity and without side-effects. The agent comprises a proanthocyanidin polymer composition illustrated in the following the general formula (1), 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1  is hydrogen or hydroxyl, R 2  is hydroxyl, R 3  is hydrogen when R 1  is either hydrogen or hydroxyl, but R 3  is possibly hydroxyl when R 1  is either hydrogen or hydroxyl to the extent that both R 1  and R 3  being hydroxyl is at most 40 percents in the proanthocyanidin polymer composition 
             said units of flavan-3-ol being bonded each other in any one of three patterns as follows; 
             (i) a bond between carbon at the position 4 and carbon at the position 8, 
             (ii) a bond between carbon at the position 4 and carbon at the position 6, 
             (iii) a bond between carbon at the position 4 and carbon at the position 8, and between carbon at the position 2 and oxygen at the position 7.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an agent for inhibiting production ofhepatitis C virus. In particular, this invention also relates to use ofproanthocyanidin.

2. Related Background Art

Hepatitis C virus (hereinafter referred to as HCV) has been found as amajor causal virus of non-A non-B hepatic inflammation after bloodtransfusion. HCV is a single-strand RNA virus having an envelope andbelongs to Hepacivirus of Flaviviridae.

In some cases, HCV carriers progress directly to chronic hepatitiswithout acute hepatitis and then to cirrhosis after many years. About 50percents of HCV carriers go onto the development of hepatocellularcarcinoma. It is said that main cause of such development is persistentinfection with HCV. The persistent infection tends to occur even inadults who were infected with HCV after establishing immune system. Evenin case of a patient whose site or sites of hepatocellular carcinoma hasbeen completely removed by surgery, the patient still remains at highrisk of recurrence, result of the persistent infection repeatedlyoccurred in unremoved sites.

Japanese Patent Application Laid-Open No. 2007-119398 disclosed that theprocess materials of blueberry leaves are effective for inhibitingproduction of HCV. The publication has, however, no disclosure as to anyeffective chemical substance contained in blueberry leaves.

It is reported in Japanese Patent Application Laid-Open No. 2005-239581that Pycnogenol or proanthocyanidin has anti-virus activities and isuseful to improve the quality of life in carriers of HCV. In thispublication, the target virus effective of Pycnogenol is myocarditisvirus, but it is not HCV. The inhibition of HCV production, which isunlike myocarditis virus, is not able to evaluate by any model animalexcept chimpanzee. In view of this, there is no explicit description inthe publication about the inhibition of HCV production byproanthocyanidin.

WO2004/047847 refers to an antiviral action of proanthocyanidin andsuggests that the proanthocyanidin might be usable for treating hepaticinflammation. Nevertheless, the antiviral action in the publication isagainst West Nile virus of Flaviviridae. Although HCV belongs to samefamily, it differs from the West Nile virus in taxonomic genus and thesite of infection. The publication has no suggestion as to inhibitoryactivity of proanthocyanidin for production of HCV.

U.S. Pat. No. 5,211,944 disclosed that a proanthocyanidin polymercomposition having 2 to 11 flavonoid units is specifically useful fortreating respiratory virus infection such as respiratory syncytialvirus, influenza virus and parainfluenza virus infection, but therespiratory virus is quite different from HCV. So, the publication dosenot drop any hint about possible activity of proanthocyanidin againstHCV production.

These days, the interferon therapy is only effective method forinhibiting production or proliferation of HCV, or eliminating HCV. Evenso, around 50 percents of the HCV carriers still remain at risk foronset of hepatocellular carcinoma due to failure to clean HCVcompletely. It is also suggested that interferon should be administeredtogether with ribavirin to enhance disappearance rate of HCV. Further,continuous dosing of interferon in a low dose has been introduced toinhibit progress of hepatitis C disease and then to retard eventuallythe onset of hepatocellular carcinoma.

In each case, it is unavoidable to leave some patients who cannotreceive the continuous interferon therapy for a long period, because ofits strong side-effects such as dehairing, decreased appetite,thrombopenia, depression of white blood cell and so on (See; Shiro Iino,Medical Digest, vol. 46, no. 6, pp. 19-22, 1997).

An object of this invention is to provide an agent for effectivelyinhibiting production of HCV with fewer side-effects.

Another object of this invention is to provide an agent for inhibitingonset, progress and treating of hepatic disease arisen from HCV.

Further object of this invention is to provide dietary supplements forinhibiting onset or progress of hepatic disease arisen from HCV.

SUMMARY OF THE INVENTION

We, inventors had already found that blueberry leaves have strongsuppressive activity against production or proliferation of HCV (See;Japanese Patent Application Laid-Open No. 2007-119398). At that time, itwas unclear what effective chemical substance contained in blueberryleaf is. As a result of continuing research on this matter, we havesuccessfully identified that the proanthocyanidin polymer compositionwith specific chemical formula contained in blueberry leaves has thestrong inhibitory activity of HCV production. And then such a findinghas led us to this invention.

An aspect of this invention resides in an agent for inhibitingproduction of HCV comprising a proanthocyanidin polymer composition. Theproanthocyanidin polymer composition has a structure in whichflavan-3-ols illustrated in the general formula (1) are bound each otherthrough any of the bond pattern (i), (ii) and (iii) mentioned below.Among the general formula (1), the proportion of flavan-3-ol in whichboth R₁ and R₃ are hydroxyl is at most 40 percents.

(i) the bond between carbon at the position 4 and carbon at the position8(ii) the bond between carbon at the position 4 and carbon at theposition 6(iii) the bond between carbon at the position 4 and carbon at theposition 8, and carbon at the position 2 and oxygen at the position 7

In the formula (1), R₁ is hydrogen or hydroxyl, R₂ is hydroxyl, R₃ ishydrogen when R₁ is either hydrogen or hydroxyl, but R₃ is possiblyhydroxyl when R₁ is either hydrogen or hydroxyl to the extent that bothR₁ and R₃ being hydroxyl is at most 40 percents in the proanthocyanidinpolymer composition, and R₄ is hydrogen or a univalent organic acidgroup.

In this invention, the agent for inhibiting HCV production comprisingthe proanthocyanidin with the structure aforementioned has advantages offewer side-effects, dosing over a long period of time and effectiveanti-HCV production activity.

In the agent containing proanthocyanidin, R₄ in the general formula (1)is preferably a gallate group allowed to have substituent groups.

Further, in the HCV production inhibitor of this invention, the ratio ofthe concentration of proanthocyanidin polymer composition inhibiting 50percents of HCV virus proliferation (IC50) divided by the concentrationof proanthocyanidin polymer composition inhibiting 50 percents of cellproliferation (CC50) is desirably at most one tenth. Such an inhibitingagent of HCV production has fewer side-effects and can be dosed for along period of time.

The HCV production inhibitor of this invention has the structure inwhich proanthocyanidin polymer composition has preferably flavan-3-olunit of the general formula (1) bound each other in any one of the bondpattern (i), (ii) or (iii).

Another aspect of this invention resides in an agent or a prodrug fortreating the hepatic disease arisen from HCV.

Another aspect of this invention resides in an agent or a prodrug forinhibiting onset or progress of the hepatic disease arisen from HCV.

Further aspect of this invention resides in a dietary supplementincluding food or drink for inhibiting onset, progress or treating ofthe hepatic disease arisen from HCV.

According to this invention, the agent for inhibiting production of HCVhas advantages of significant anti-HCV activities with fewerside-effects and a long dosing period of time.

The inhibiting agent of HCV production comprising the proanthocyanidinhas less cytotoxicity in addition to higher anti-HCV repliconproliferation in vitro. It is, therefore, useful for alternativemedicines or dietary supplements instead of interferon. The agent hasalso advantages to be able to dose continuously for a longer period oftime, owing to fewer side-effects compared with interferon. In case ofthe patients having cirrhosis or hepatocellular carcinoma, numbers ofplatelets or leucocytes are so small that they would be unable toreceive continuously the interferon therapy. The therapy using the agentof this invention can, therefore, become an effective alternativetherapy for interferon. The proanthocyanidin is useful for a functionaldietary supplement including food or drink as well.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows HCV replication suppressive activity of PAC from blueberryleaves in replicon cells.

FIG. 2A shows HPLC chromatogram of methanol extract from blueberryleaves.

FIG. 2B shows the replication suppressive activity of fractionation byHPLC according to FIG. 2A.

FIG. 3A shows HPLC chromatogram of 1st fractionation (LC1) of methanolextract from blueberry leaves.

FIG. 3B shows the replication suppressive activity of LC1 by HPLCaccording to FIG. 3A.

FIG. 4A shows HPLC chromatogram of 2nd fractionation (LC2) of LC1.

FIG. 4B shows the replication suppressive activity of LC2 by HPLCaccording to FIG. 4A.

FIG. 5A shows HPLC chromatogram of 3rd fractionation (LC3) of LC2.

FIG. 5B shows the replication suppressive activity of LC3 by HPLCaccording to FIG. 5A.

FIG. 6 shows elemental composition analysis of LC3 (purified fractionfrom blueberry leaves) by electron probe micro-analysis (EPMA)

FIG. 7 shows mass spectrum of LC3 (purified fraction from blueberryleaves) by LC/APCI-MS-IT-TOF

FIG. 8A shows MS/MS spectrum of cyanidin preparation by LC/ESI-MS.

FIG. 8A shows MS/MS spectrum of LC3 (purified fraction from blueberryleaves) by LC/ESI-MS.

FIG. 9 shows thiolysis reaction pattern of PAC reacted withbenzylmercaptan under acidic condition.

FIG. 10 shows reverse-phased HPLC chromatogram of thiolysate from LC3(purified fraction from blueberry leaves).

FIG. 11 shows the relationship with mean degree of polymerization (mDP)and specific activity (1/IC50) of Sephadex LH-20 fractionation fromblueberry leaves.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to the drawings according to need, the preferred embodimentsof this invention are described in detail, but does not limit thisinvention in scope.

The inhibitory agent for production of HCV in this invention comprises aproanthocyanidin polymer composition (herein after referred to as PAC).

The PAC in this invention is a polymer composition of matter wherein themonomer unit in the general formula (1) is combined each other in threebond patterns below. The number of monomer unit defining thepolymerization degree of PAC is at least 3, preferably at least 5, morepreferably 5 to 10. The typical bond patterns of the monomer unit aredescribed bellow.

(i) the bond between carbon at the position 4 and carbon at the position6(ii) the bond between carbon at the position 4 and carbon at theposition 8(iii) the bond between carbon at the position 4 and carbon at theposition 6, and carbon at the position 2 and oxygen at the position 7

General Formula (1);

In this formula (1), R₁ is hydrogen or hydroxyl, R₂ is hydroxyl, R₃ ishydrogen when R₁ is either hydrogen or hydroxyl, but R₃ is possiblyhydroxyl when R₁ is either hydrogen or hydroxyl to the extent that bothR₁ and R₃ being hydroxyl is at most 40 percents in the proanthocyanidinpolymer composition, and R₄ is hydrogen or a univalent organic grouprespectively.

As the univalent organic group at the position R₄, there can be listed agallate group, an organic acid residue, and a sugar residue. The organicacid may be p-coumarin acid, caffeic acid, ferulic acid, sinapic acid,p-hydroxyl benzoic acid, gallic acid, acetic acid, oxalic acid, malonicacid, succinic acid, malic acid and so on.

R₄ may also be glycoside residue in which sugars are bound with organicacid residue or gallate groups. The sugars are bound with a phenolichydroxyl of the gallate groups or the organic acid groups through esterbonds at any position. For instance, there are hexose residues ofpyranose type and pentose residues of furanose type. The sugars may bemonosaccharide including glucose, galactose, rhamnose, xylose andarabinose, and either disaccharide or trisaccharide.

Among them, R₄ is preferably a gallate group permitted to havesubstituent groups consisting of a gallate group or a gallate group withsubstituent. The gallate group with substituent includes a gallate groupwith which sugar are bound.

Further, each hydroxyl of A-ring at the position 5 and/or the position 7may be replaced by modified groups such as a gallate group, sugar,organic acid and the like.

Among all flavan-3-ol units forming the PAC polymer composition, theproportion of flavan-3-ol of which both R₁ and R₃ are hydroxyl is atmost 40 percents. In case the proportion is more than 40 percents, it isinsufficient to suppress production of HCV. The proportion can bemeasured by thiol cleavage method.

For example, one of the PAC of this invention cab be illustrated in thegeneral formula (2), wherein flavan-3-ol units are bound each other atthe bond pattern (i).

Further, there may be a PAC illustrated in the general formula (3),wherein flavan-3-ol units are bound each other at the bond pattern(iii).

In the general formula (1), R₁ and R₃ are hydrogen or hydroxyl, R₂ ishydroxyl, R₄ is oxygen or a univalent organic group, provided that theproportion of both R₁ and R₃ being hydroxyl is at most 40 percents. Onlysuch a PAC polymer composition can express suppressive activity of theHCV replication in this invention. While the PAC polymer compositionwherein the proportion of both R₁ and R₃ being hydroxyl is more than 40percents cannot meet the criterion level of the suppressive activity ofHCV replication in this invention.

Although the position 5 and/or 7 of A-ring in the monomer unit of thegeneral formula (1) is hydroxyl, at least one of them may be a univalentmodified group bound therewith. Typical modified groups may be a gallategroup, sugar or organic acid.

In this invention, the anti-HCV production activity is actually anactivity for inhibiting the replication of HCV replicon cell in vitro,and can be expressed as anti-HCV production activity value (IC50; μg/ml)or the ratio between the anti-HCV production activity value (IC50;μg/ml) and the cytotoxicity value (CC50; μg/ml). It is desirable thatthe anti-HCV production activity value (IC50; μg/ml) is at most 1.0;μg/ml in terms of the IC50 of interferon. While the ratio ofcytotoxicity activity value (CC50; μg/ml) divided by anti-HCV productionactivity value (IC50; μg/ml) is at least 10 in terms of effectiveness asmedicine (See; Naoya Sakamoto et al. Japan clinic, vol. 62, p. 116-120,2004). The effect of PAC in this invention can be evaluated with theratio.

PAC is a kind of the condensed tannin contained in various plants, andcan be treated by acids to give cyanidin, delphinidin, pelargonidin,aurantinidin, luteolinidin, peonidin, malvidin, petunidin, europinidin,rosinidin, hirsutidin, apigeninidin and so on. As illustrated in thegeneral formula (1) and (4), the PAC belongs mainly to catechin class,namely, polyphenol groups consisting of condensation polymer, of whichpolymerization degree of the flavan-3-ol units is at least 2. In thesegeneral formulas, R corresponds to OR₄ set forth above. Further, agallic acid is often bound with hydroxyl at the position 3 offlavan-ring through an ester-link. In other words, PAC can be said to bea generic term including various compositions of matter formed bypolymerization of the flavan unit which has hydroxyl at some bindingpositions. Among the units constituting the polymer, the rear anchor isreferred to as ‘terminal unit’ and the other units ‘extension unit’. Apolymerization degree of the units extends over the wide range fromdimmer to polymers having 100 units or more.

Some polymers in which specified monomer is polymerized are calledtrivial names. The typical trivial names defined or classified dependingon numbers of hydroxyl in B-ring of the flavan units are properalgonidin(4′-OH), procyanidin (3′-OH, 4-OH) or prodelphinidin (3′-OH, 4′-OH,5′-OH). In addition, there are proguibourtinidin, profisetinidin andprorobinetinidin, all of which has no hydroxyl at position 5 of A-ringof the flavan units. Depending on whether hydroxyl would be at thespecific binding position of flavan units, there are given such atrivial name as proteracacidin, promelacacidin, proluteolinidin and soon.

In this invention, the PAC comprises proanthocyanidin, properalgonidinand/or prodelphinidin. The constituent units of procyanidin may beeither one or more of same or different groups, and at least oneselected from the group consisting of catechin, epicatechin, catechingallate and epicatechin gallate. The constituent units of prodelphinidincan also be either one or more of same or different groups, and at leastone selected from the group consisting of gallocatechin,epigallocatechin, gallocatechin gallate and epigarocatechin gallate.

In case R₄ of the PAC in the general formula (1) is residues of theunivalent organic acid, such as a gallate group, residue of sugar,residue of organic acid, wherein term ‘residue’ means a univalent groupor groups caused by losing atom or group in the molecular. As theorganic acid in the organic acid residues, there are p-coumaric acid,caffeic acid, ferulic acid, sinapic acid, p-hydroxyl benzoic acid,gallic acid, acetic acid, oxalic acid, malonic acid, succinic acid, andmalic acid and so on.

Sugar in the sugar residue may be bound with the hydroxyl of phenol inany binding position thereof through an ester bond to modify. Forinstance, the residues constituting glycoside may be hexose residue ofpyranose form and pentose residue of furanose form. As concrete exampleof form of saccharide, there is monosaccharide including glucose,galactose, rhamnose, xylose, arabinose etc., and other disaccharide ortrisaccharide.

The main binding positions among units constituting the PAC are (i) (ii)or (iii) set forth above. As practical embodiment, there are A-type bondconsisting of both the bond of carbons at the position 4 and 8, and thebond of carbon at the position 2 and oxygen at the position 7, andB-type bond consisting of either the bond of carbons at the position 4and 6, or bond of carbon at the position 4 and 8. In addition to these,there are various stereoisomeres with the combination of these bondforms.

The PAC as illustrated in the general formula (3) and (4) has A-typebond and B-type bond. According to the findings of the inventors,however, the difference in the extension of A-type bond and B-type bond,or the mixture ratio therebetween does not affect the anti-HCVproduction activity of the PAC. In the PAC extracted from blueberry,both of the polymerization degree and the proportion of hydroxyl inB-ring in the PAC polymer composition must meet the criterion of thisinvention, though the B-type bond is dominant.

According to the differences in binding positions on the rings, thereare various PAC polymer compositions, such as steric conformation ofsubstituent groups in flavan unit and binding order among a variety offlavan units. The chemical names of the PAC in this invention are listedas follows. The letters in parentheses express chemical formula. Thisinvention may encompass PACs of higher polymerization degrees, which arepolycondensated with constituent units such as catechin and epicatechin,or with other proanthocyanidin.

Proanthocyanidin PZ 5 (C75H62O31)

Proanthocyanidin BP 1 (Unspecified)

Proanthocyanidin RP 4 (C129H106O67)

Proanthocyanidin RP 3 (C136H120O70)

Proanthocyanidin CS 4 (C136H120O70)

Proanthocyanidin CS 3 (C127H128O69)

Proanthocyanidin CS 2 (C113H110O62)

Proanthocyanidin CS 1 (C121H118O65)

Proanthocyanidin RP 2 (C120H114O64)

Proanthocyanidin RP 1 (C125H130O69)

Proanthocyanidin T 4 (C128H122O65)

Proanthocyanidin T 3 (C105H102O59)

Proanthocyanidin T 2 (C67H54O29)

Proanthocyanidin T 1 (C87H72O43)

Proanthocyanidin C 1 (C45H38O18)

In this invention, the mean polymerization degree of the PAC ispreferably at least 5 in terms of anti-HCV production activity as shownin FIG. 11. However, if a PAC polymer composition contains at least 3 ofmonomer unit of the general formula (1) in the mean polymerizationdegree; its anti-HCV replicon production activity value is at most 1.0μg/ml; the ratio of cytotoxicity activity value (CC50) divided byanti-HCV production activity value (IC50) is at least 10, such PACpolymer compositions are covered in this invention.

In this invention, the mean polymerization degree of the PAC means theaverage number of flavan-3-ol units bound each other with the bondpattern (i), (ii) and (iii). The number of flavan units can bedetermined by the method described in the example 1.

The PAC may be either synthetic product or natural product. The naturalproduct can be derived from some plants containing polyphenol. It isknown that some colored fruits and vegetables are high content ofpolyphenol. There can be illustrated by examples of blueberry leaves,grape seeds, taro, bilberry, elderberry, plum, blackberry, strawberry,redcurrant, blackcurrant, cranberry, cherry, raspberry, currant, ahibiscus flower, green pepper, beans, peas, soybean skin, a red cabbage,a purple corn, a purple sweet potato, herbs, fern, nuts, bark etc.Particularly, the blueberry leaves, the grape seeds and taro are veryeffective for the inhibitory activity of HCV production. The skin ofTaro contains a large amount of proanthocyanidin. It may be practical touse the skin of Taro, in spite of the unsuitable part for eating.

Various parts of a plant or its processed good are also useful for thestarting material of this invention. The usable starting material is,for example, leaves, petals, calyx, flowers, leafstalks, fresh tops,roots, stems, seeds, pods, rhizomes, barks, cambiums, lumbers,mycocecidium, fruits, tree saps, resin, a peel of grape, apple, onion,avocado and citrus; pomas of apple, wine, grain hull, straw and hay; thelumps from oily seeds derived from olive, oilseed rape and canola; andthe extracts from other oily crops etc.

The most preferable starting material is blueberry shown in theexample 1. The blueberry belonging to Cyanococcus of Vaccinium ofEricaceae is a deciduous or evergreen fruit tree, which is a low tree ora low bushes originally comes from America. The Usable blueberries inthis invention are without reference to kinds and native localitiesthereof and are limited to the listed above. The blueberry is generallyclassified into around six kinds, among which the routine use ofhorticulture is said to be three kinds below.

(1) Highbush blueberry (Vaccinium corymbosum L.); O'Mear, sharpblue,Flordablue, Reveille, Spartan, Darrow, Duke, Berkeley, Harrison etc.

(2) Rabbiteye blueberry (V. Firgatum Aiton); Woodard, Garden blue,Tifblue, Homebell, Myers etc.

(3) Lowbush blueberry (V. angustifolium Aiton, V. myrtilloides Michaux);Chignecto, Brunswick, Blomidon etc.

Rabbiteye blueberry is most useful staring material among them listedabove.

The PAC as effective ingredient in this invention can be produced bycombination of following steps depending on intended use. (1)Pretreatment step: First of all, blueberry leaves are washed with waterand filtered to remove unusable miscellaneous things therefrom. Theunwanted components such as cellulose, chlorophyll etc. in the crudematerial after washing are distilled away with organic solvent. As theorganic solvent, chloroform, hexane, acetone etc. and the combinationthereof can be properly used. The distillate may be supplied to nextstep directly or after pulverizing with or without drying. Extracted orsqueezed juice from the crude material can also be supplied as well.Before supplying next step, the extracted or squeezed juice may becondensed, dried and pulverized into powder.

Any conventional dryer can be used in this invention, so long aschanging nature of chemicals in the crude material. For instance, avacuum-freeze dryer, a hot air dryer, a far-infrared radiation dryer, areduced-pressure dryer, a micro-wave reduced-pressure dryer, asuper-heated steam dryer etc. can be used. Among them, the vacuum-freezedryer is more useful, because of having few effects on the nature of thechemicals. The Vacuum-freeze dryer should be used under the designatedcondition depending on the state of leaves to be processed. For example,in case of fresh leaves, it is desirable that freezing temperature isfrom −30 to −20° C.; drying temperature from −30 to 30° C.; and dryingperiod 15 to 24 hours.

(2) Extract step: The processed material in the former steps is thenextracted with a solvent or solvents through single or multiple steps.In the extract process, the solvents may be used either alone or thecombination thereof. For example, water or polar solvent compatible withwater can be preferably used. As the solvent compatible with water,there are lower alkyl alcohol with numbers of carbon 1 to 4, such asmethanol, ethanol, propanol, butanol etc. and polyol such as ethyleneglycol, butylene glycol, propylene glycol, glycerin etc. Among them,lower alkyl alcohol such as methanol or ethanol may be practical interms of safety.

Other organic solvent, such as acetone, diethyl ether, dioxane,acetonitrile, ethyl acetate, xylene, chloroform, toluene, hexane etc.can also be used. These solvents may be used in either single or thecombination thereof, such as the combination of water and polar solventcompatible with water. For instance, the mixed solvent of acetone andethyl ether can be used preferably in the ratio of 1:1 by v/v. It isgeneral to use the mixture of water and polar solvent as the mixedaqueous solvent. As aqueous alkyl alcohol, aqueous methanol and ethanolmay be preferably used. The alcohol content in the aqueous alcohol is inthe range of 5 to 90 percents, preferably 30 to 90 percents, morepreferably 50 to 90 percents. It is recommended to use mixed alcoholsolvent containing water or acetone in this invention.

In the extraction process, the processed material in above steps isimmersed in cool or warm solvent. It is usually extracted under warmingand agitating and then filtrated to give extracted liquid. For example,the extraction may be preferably carried out using an aqueous alcoholsolvent containing 80 volume percents of alcohol at room temperature andfor immersion periods of 30 to 60 minutes depending on the extractingtemperature. The extraction can be done by percolation method too.

(3) Purification step: The impure solid materials can be removed fromthe obtained extract by filtration or centrifugal separation to givepure filtrate, if needed. The filtrate may be supplied to the next stepdirectly, or after partially condensed by distillation or dried.Further, these condensed or dried materials can be further purified bythe method such as column method or fractionation method with solvent(See; WO2000-64883).

Taro (Colocasia esculenta Schott) usable in this invention as one of thematerial of the PAC belongs to Colocasia, Araceae, originated from theSouth Asia and is widely cultivated in the many varieties over theworld. The root stocks and leaf stems have been mainly utilized forfoodstuffs so far. Among araceae, there is konjac (Amorphophalluskonjac), which can be used as foodstuff after processing. In addition,there are several varieties in araceae for appreciation too. It is knownthat the root stock of Araceae plants contains mucin, galactan andpotassium as effective ingredients. It is also reported that galactanhas activity of brain cell and immunity. Potassium prevents high bloodpressure due to removing sodium. But there have been no report onanti-HCV production activity of the root stock of Araceae plants untilnow.

The root stocks of Araceae plant are generally used after processing inthis invention to realize effective anti-HCV production. As typicalprocessing method of the root stocks, there are pulverization, driedpulverization, squeezation and solvent extraction.

In general, the root stocks are pulverized after drying or slicingbefore drying. The root stocks of Araceae can be dried according to thedrying method of blueberry leaves set forth above. The extractionprocess and purification process can also be carried out by the same wayas blueberry leaves.

The PAC in this invention can be prepared into various formulations suchas extracted solution, condensed solution, paste, and dried or semidriedpowder. The purified PAC is understandably higher anti-HVC productionactivity compared with crude extracts.

There is no appropriate evaluation system for anti-HCV productionactivity in vivo except chimera mouse, because HCV dose not infect allanimals other than primates. In this invention, HCV replicon cell asknown in vitro evaluation system of HCV replication (Lohmann V. et al.,Science Vol. 285, p. 110-113, (1999)) is employed as alternative vivoevaluation method. HCV replicon cell comprises a structural proteintranslation region with core and envelop constituting virus particle,and non-structural protein translation region having function of virusgenome replication etc. The HCV replicon cell may be made from thenon-structural region. The replication of HCV-RNA in this system ismeasured as replication numbers of whole HCV-RNA infected with HCV.

The effective mean degree of polymerization (mDP) of the flavan unit ingeneral formula (1) is at least 3 in integer value. As shown in table 1,no anti-HCV production activity is found in catechin (DP=1) and catechindimmer (DP=2). These findings suggest that other epicatechin, afzelechinand epiafzelechin would be few activity of anti-HCV production too.

TABLE 1 The activity of inhibiting replicon production HCV ReplicationSuppressive Activity of Catechin Monomer and Epicatechin DimerReplication suppressive Cytotoxicity Ratio activity IC50(μg/ml)CC50(μg/ml) CC50/IC50 Catechin 16.18 100.4 6.2 Epicatechin 27.32 113.84.2 Procyanidin B2 >25.0 >25.0 —

This invention encompasses PAC polymer composition wherein the positionR₂ of flavan unit in the general formula (1) is hydroxyl; the positionR₁ and the position R₃ are hydrogen or hydroxyl; and the position R₄ ishydrogen or modified groups, so far as the proportion of both R₁ and R₃being hydroxyl is at most 40 percents of whole polymer composition. Theanti-HCV production activity of gallocatechin and epigallocatechin socalled generally prodelphinidin wherein all the position R₁, R₂ and R₃in B-ring of the flavan unit are hydroxyl is likely quantity dependantinverse proportion as illustrated in the table 2. For instance, in casethe proportion of the flavan units such as croton sap exceeds 40percents of whole polymer composition, such polymer composition becomesstronger in cytotoxicity and less than or equivalent to 10 in the ratiobetween IC50 and CC50 as shown in the table 3 of the example 1.Accordingly, the polymer composition is unusable in practice as theinhibitor of HCV production in this invention.

The PAC of this invention may be produced chemically or biologically.The PAC is also purified directly from the natural material such asplants, provided that its structure and function fall within the scopedefined in this invention.

The PAC may be supplied to end users in various formulations such asextracted solution, condensed solution, and dried or semidried powderetc. depending on their request.

The inhibitory agents of HCV production in this invention may beavailable directly, or as either prodrug or formulated medicine. The PACmay be administered to persons infected with HCV, in the form of eitherpolymer composition alone as shown in the general formula (1), ortogether with medically acceptable salts, hydrates and solvates. Ingeneral, it seems to be rather effective to dose a drug comprising thePAC and pharmacologically and pharmaceutically acceptable additives.

As the pharmacologically and pharmaceutically acceptable additives,there may be diluent, disintegrant, auxiliary disintegrant, binder,lubricant, coating, pigment, base agent, solvent, auxiliary solvent,isotonic agent, pH regulator, stabilizer, injection agent, adhesive andso on.

When using the PAC as a drug, it is desirable to administrate in everydosing unit. As such dosing manner, there are oral administration,enteral administration, intravenous administration, local administrationsuch as dermal administration, and interstitial administration such assubcutaneous administration, intramuscular administration etc.

As solid dosage formulation for oral administration, there may betablet, ball, capsule, subtle granule or pill. In addition, there may beacceptable emulsion, suspension, and syrups as liquid dosage formulationfor oral administration. When formulating drugs, lubricant, colorant,flavoring agent, and/or pH regulator can be added to the base agentincluding the PAC. The tablet may be coated with conventional sugarcoating, gelatin coating, enteric coating tablet, film coating, doublecoating and multiple coating, if required. As the additives, there maybe used diluents such as lactose, sucrose, sodium chloride, glucose,urea, starch, calcium carbonate, kaolin, crystalline cellulose, hydratedsilica etc.; binders such as water, ethanol, propanol, sirup, glucosesolution, starch solution, gelatin solution, carboxymethyl cellulose,shellac, methyl cellulose, potassium phosphate, polyvinyl pyrolidoneetc.; disintegrants such as dried starch, sodium argininate, agarpowder, laminarin powder, sodium hydrogen carbonate, calcium carbonate,polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate,stearic acid monoglyceride, starch, lactose etc.; disintegrantsuppressing agent such as sucrose, stearin, cacao butter, hydrogenatedoil etc.; adsorption promoter such as quaternary ammonium salts, sodiumlauryl sulfate etc.; humectants such as glycerin, starch etc.;absorbents such as starch, lactose, kaolin, bentonite, colloidalhydrated silica etc.; and lubricants such as purified talc, stearate,boric acid powder etc.

As the enteral administration, there may be the use of injectableproducts. When preparing the injectable products, it can be made informulation of nontoxic solution, suspension or emulsion preferablysterilized and adjusted to the isotonicity nearly equal to blood. Thesesuspension and emulsion can be prepared by using water; ethanol,propylene glycol, ethoxyisostearyl alcohol, polyoxyisosteayl alcoholetc; and/or polyoxyethylene sorbitan fatty acid ester. Further, therecan be added sufficient amounts of salt, glucose and/or glycerin etc soas to obtain isotonic solution. In addition, usual solubilizing agents,buffering agents, pH regulating agents, soothing agents and the like canalso be used. The injectable product can be administered under skin,into muscle or vein.

For local administration, there may be topical solution, cream, powder,paste, gellant and ointment. These are prepared by combining a givenquantity of the effective ingredients including the PAC, and itspharmacologically and pharmaceutically acceptable prodrug or salts,together with additive usable for topical medicine such as fragrance,colorant, filler, surfactant, humectant, emollient, gellant, carrier,preserving agent and/or stabilizer.

For the formulation of interstitial administration, a suppository istypical. When preparing the suppository, there can be used higheralcohol; higher ester such as myristicin palmitate ester, polyethyleneglycol, cacao butter, gelatin, semi synthetic glyceride and theirmixture as substrates with low melting point. The suppository can beformulated by adding the PAC, its prodrug and their medically acceptablesalt to these substrates.

When ministering the PAC or the inhibitory agent of HCV productioncontaining its prodrug with their medically acceptable salt to patientsneeded thereof, the effective dosing quantity should be determined inview of condition of each patient of which condition are, for instance,a time of life, weight, the route of administration, the nature or levelof disease etc. This invention suggests that the effective dosingquantities may be within the range of 1 to 2000 mg per a day for anadult human. But, according to clinical condition of a patient, theeffective dosing quantities may be less than or larger than thesuggested range. When dosing larger quantities than the suggested range,it is desirable to dose fractionally at several times per a day. Theagent of this invention can also be adopted for preventing or inhibitingonset or progress of hepatic inflammation and cirrhosis, or treatinghepatitis diseases arisen from HCV, in addition to the inhibition of HCVproduction that is the primary activity of this invention.

In case of pharmaceutical agent, the effective quantity ofadministration to a patient can not be determined strictly owing tovariable dependant on the expected effect of treatment, administrationmethod or its root, dosing periods, gender of a patient, and othercondition of disease. Typically, the dosing quantities of the anti-HCVinhibitor including crude or purified production of this invention maybe selected from the range about 100 to 1000 mg per a dose for theweight of 60 kg in a human patient as reduced weight of the purified PACof this invention. The content of the PAC in the agent may be selectedfrom the range of 0.1 to 99.5 weight percents, preferably 0.5 to 90weight percents, though the content is needed not to specifically limit,because of variable dependant on the dosing quantities. Whenevernecessary, the agent of this invention can be dosed. But, it iseffective to dose in acute or chronic stage of hepatitis inflammation orstage of cirrhosis during which the state of hepatic disease is inprogress.

Further, the PAC of this invention may be usable for dietary supplementssuch as food or drink. The supplements can be prepared in solid, liquidsolution, emulsion and so on, and shaped into various forms such astablet, ball, capsule, granule, powder, pastille, cake, drink etc.According to need, pharmacologically, pharmaceutically and dietaryacceptable additives can be added. The additives include other variousmedical substances or functional ingredients such as vitamin, otherminor components and the like.

EXAMPLES

Following examples illustrate more detail embodiments of this inventionwithout, however, limiting the invention in any way. In the workingexample, percent (%) means weight percent (w/w %), unless otherwisestated.

[Experimental Procedure]

In the working example, the HCV replication suppressive activity test(replicon assay) and cytotoxicity test were carried out as follows.

The HCV-replicon cell line was established by transducing sub-genomicRNA into human hepatoma Huh-7 cell cytoplasm. The sub-genomic RNA wasprepared by replacing the translated region of HCV genome RNA structuralproteins with firefly luciferase gene, internal ribosome entry site(IRES) of the encephalomyocarditis virus and neomycin phosphotransferasegene. The cell line obtained was used for assessment of production ofHCV replicon RNA. The production quantity of HCV replicon RNA wasmeasured by the luciferase assay method. (Lohmann et al., Science 285,(1999) 110; Sakamoto et al. Nat. Chem. Biol. 1, (2005) 333)

Anti-Replicon Activity Test (Replicon Assay)

In order to determine replication quantity of HCV-RNA, luciferase genesfrom firefly were transduced into HCV-RNA as a reporter gene. The genetransduction was carried out according to the method of Krieger et al.(See; Krieger et al. J. virol. 75, (2001) 4614) in such manner thatluciferase genes were fused together neomycin-resistant genes beneaththe IRES of HCV gene.

With this HCV subgenome, the replication efficiency of HCV could beestimated by measuring luciferase activity in the HCV replicon cells.The HCV replicon cells used were routinely grown in DMEM (Gibco cat. No.10569-010) supplemented with Glutamax (Invitrogen), 10% fetal bovineserum (FBS), 1% penicillin/streptomycin (Invitrogen) and 500 μg/ml G418(Invitrogen). The cells were maintained at 37° C. in humidifiedatmosphere containing 5% CO2. For the HCV replication assay, thereplicon cells in DMEM supplemented with Glutamax and 5% FBS were seededin a 96-well plate so as to fill every well with 5000 cells, andincubated for 24 hours.

The test was carried out in such a way that the HCV replicon cells werecultured for 72 hours after adding test samples in differentconcentrations. The Quantity of the luciferase activity was determinedby the Steady-Glo Luciferase Assay System (Promega) according to themanufacture's instructions and the luminescence was measured by DTX 800Multimode Detector (Beckman). In this test, the replication inhibitoryactivity is expressed as a concentration of samples required forinhibiting 50 percents of the replication of HCV replicon cell (IC50).Specific activity is a reciprocal number of IC50 (1/IC50). Totalactivity is calculated by multiplying the yield weight of the HCVreplicon cell by specific activity.

Cytotoxicity Test

The cytotoxicity of the samples was measured by Cell Counting Kit-8(CCK-8; Dojindo Molecular Technologies) according to the manufacture'sinstructions. Briefly, 10 μl/a well of CCK-8 reagent were added to theHCV replicon cells cultured in a 96-well plate and then incubated at 37°C. for 60 min. The absorbance of each well was measured at 450 nm with areference wavelength at 650 nm using an Emax Precision microplate reader(Molecular Devices Inc.). Cell viability was calculated as relativeindex of control cells and effects of samples and was expressed as theconcentration of samples required for 50% cytotoxicity of the cells(CC50).

FIG. 1 illustrates the results of a PAC sample from blueberry leaves inreplicon assay. The IC50 value showing the replication suppressiveactivity was 0.56 μg/ml and the CC50 value showing the cytotoxicity was16.26 μg/ml. The IC50 was less than or equal to 1.0 μg/ml and the ratio,which was calculated by dividing CC50 by IC50, was larger than or equalto 10. In conclusion, the PAC sample has the inhibitory activity of HCVreplication.

Example 1 Identification of Effective Compounds for Suppressing HCVReplication Contained in the Blueberry Leaves

1. Extraction and Liquid-Liquid Distribution

One gram of the lyophilized powder, which made from the leaves ofrabbit-eye blueberry (Vaccinium virgatum Aiton), was extracted with 100ml of methanol at room temperature under shaking for 15 min, and thesupernatant of the resultant extract was passed through filter paper(Toyo filter paper No. 2). The methanol extract was further extractedwith 100 ml of chloroform, followed by the resultant precipitate andsupernatant were collected. The precipitate was dissolved in methanol,concentrated in vacuo and lyophilized (CMW-ppt: 63.7 mg). Thesupernatant was mixed with 150 ml of distilled water and methanol toperform a liquid-liquid extraction, and the water layer was collectedand mixed with 150 ml of chloroform so as to repeat the chloroformextraction. The water layer was concentrated and lyophilized (CMW-W:284.2 mg). The chloroform layer was also concentrated and lyophilized(CMW-C, 56.3 mg).

The suppressing activity of HCV replication was not detected from theCMW-C fraction in replicon cell assay, but detected from the CMW-ppt andthe CMW-W. In following purification, the CMW-W with a large recoveredamount in weight and a large amount of total activity was used.

2. The Fractionation by HPLC (Confirmation of Elution Time by HPLC)

To separate the components in the CMW-W fraction possessing HCVreplication inhibitory activity, fractionation and purification wereperformed using HPLC (Prominence System; Shimadzu) with UV detector andphotodiode array detection (PDA). The other analytical condition showsas follows.

Instrument: Shimadzu Prominence LC-20A

Column: Atlantis dC18, 4.6 mm I.D.×150 mm, 3 μm (Waters), 40° C.

Eluent: (A) 0.05% (v/v) Trifluoroacetic acid

-   -   (B) Acetonitrile

Gradient: Eluent B. 15% (0 min)-25% (12.5 min)-100% (17.5 min)-100% (25min)

Flow Rate: 0.7 ml/min

Detector: 254 nm

50 μl of the CMW-W fraction dissolved in 30 ml of methanol was injectedinto HPLC and the eluted total 26 fractions were collected during aperiod from 2.3 to 19.5 min. The replicon assay of collected eachfraction was carried out in concentration of 1%, 5% and 10%.

FIGS. 2 (A) and (B) shows HPLC chromatogram and activity of suppressedreplication for collected fraction, respectively. The data indicatedthat a fraction having strong replication inhibitory activity and elutedby around 90% of acetonitrile at 17 min contains some week inhibitoryactivity fractions same as broadly eluted fraction earlier. Thoseresults suggested the possible existence of multiple replicationinhibitors in CMW-W fraction. However, the chlorogenic acid and therutin included lots in blueberry leaves and eluted at around 6 and 11min respectively were seem not to have the inhibition activityaforementioned.

(1st Fractionation) the 1st fractionation performed under the followingcondition.

Column: Atlantis T3, 4.6 mm I.D.×150 mm, 3 μm (Waters), 40° C.

Eluent: (A) 0.05% (v/v) Trifluoroacetic acid

-   -   (B) Acetonitrile

Gradient: Eluent B. 30% (0 min)-30% (7.5 min)-100% (12.5 min)-100% (20min)

Flow Rate: 0.7 ml/min

Detector: 254 nm

100 μl of the CMW-W fraction was injected into HPLC system and theeluted 26 fractions during from 2.1 to 18 min were collected. FIG. 3shows the results of HPLC chromatogram and replicon assay in thecollected fractions.

In order to purify the most active component, the CM was separated intoisocratic condition at 30% acetonitrile and the eluate from 3.3 to 5.2min, and then the latter was collected. After repeating collection,140.2 mg as active fraction (LC1) was obtained from 440 mg of methanolextracts. The IC50 value of this fraction in the HCV replication assaywas 0.89 μg/ml and the yield of specific activity was 6-fold higher thanthat of the initial methanol extracts (TABLE 2).

(2nd Fractionation) The 2nd fractionation performed under the followingcondition.

Column: Atlantis T3, 4.6 mm I.D.×150 mm, 3 μm (Waters), 40° C.

Eluent: (A) 0.05% (v/v) Trifluoroacetic acid

-   -   (B) Acetonitrile

Gradient: Eluent B. 20% (0 min)-20% (7.5 min)-100% (12.5 min)-100% (20min)

Flow Rate: 0.7 ml/min

Detector: 254 nm

The active fraction (LC1) of 140.2 mg obtained from 1st fractionationwas dissolved in 11 ml of methanol. The solution obtained was injectedinto HPLC system. The eluted fraction was collected from 2.1 to 18 min(Total 26 fractions). FIG. 4 shows HPLC chromatogram and replicon assayof collected fractions.

In the second round HPLC, a highly active fraction was eluted from 11.9to 13.2 min and collected (LC2), yielding 24.6 mg with an IC50 value0.54 μg/ml (TABLE 2).

(3rd Fractionation) In the 3rd fractionation, the eluent B was replacedby methanol and eluted with 40-65% B linear gradient under the followingcondition.

Column: Atlantis T3, 4.6 mm I.D.×150 mm, 3 μm (Waters), 40° C.

Eluent: (A) 0.05% (v/v) Trifluoroacetic acid

-   -   (B) Methanol

Gradient: Eluent B. 40% (0 min)-65% (12.5 min)-100% (17.5 min)-100% (25min)

Flow Rate: 0.7 ml/min

Detector: 254 nm

24.6 mg of the active fraction (LC2) were dissolved in 2.5 ml ofmethanol. The LC2 methanol solution was injected into HPLC system. Theeluted fraction was collected from 2.3 to 17.5 min (total 26 fractions).FIG. 5 shows HPLC chromatogram and replicon assay of collectedfractions.

The active fraction was eluted during from 3.2 to 6.2 min and collected(LC3). The finally yielded solid material of 2.9 mg had a dark fleshcolor.

TABLE 2 shows an overall purification steps from blueberry leaves. From1000 mg of lyophilized powder of the leaves, 440 mg of methanol extractswere obtained. The IC50 value of methanol extracts was 5.47 μg/ml. Fromthe 284.2 mg of the CMW-W fraction was found out the replicationinhibitory activity wherein the IC50 value was 1.74 μg/ml; the specificactivity of the CMW-W was 3-fold greater than that of the initialextracts; and the yield of the activity exceeded 200%. These valuesresult from that the interfering substances had been removed from thetest sample.

The final fraction which was purified by repeated fractionation inreversed-phase HPLC showed a 63-fold increase in specific activitycompared with the initial methanol extracts. The CC50 value of thecytotoxicity of LC3 was 18.5 μg/ml and the ratio was 212.6 that was a16.5-fold higher ratio compared with initial extracts.

TABLE 2 Purification Steps from Blueberry Leaves Specific Total ActivityTotal Weight IC50 (1/ Purification Activity Yield (mg) (μg/ml) IC50)Factor (mg/IC50) (%) MeOH 440.0 5.47 0.18 1.00 80.44 100 Extract 284.21.74 0.57 3.14 163.33 203.05 Water Layer LC1 140.2 0.89 1.12 6.15 157.53195.84 LC2 24.6 0.54 1.85 10.13 45.56 56.63 LC3 2.9 0.087 11.49 62.8733.33 41.44

3. The Elemental Composition Analysis of Electron Probe Micro-Analysis(EPMA)

For EPMA (EPMA-1600, Shimadzu), the excitation voltage and the beamcurrent were kept at 15 kV and at 100 nA, respectively. The diameter ofthe electron beam was 50 μm and the sample was processed for carbonshadowing in advance. In order to determine the constituent elements,the purified fraction (LC3) was analyzed by EPMA. This analysisindicates that the fraction is composed of carbon, oxygen and hydrogen,but not nitrogen (FIG. 6).

4. The Constitutive Analysis of Liquid Chromatography/MassSpectrometry-Ion Trap-Time of Flight (LC/MS-IT-TOF)

Identification of the anti-HCV replication fraction (LC3) purified fromblueberry leaves was done by HPLC-MSn fragmentation analysis. An HPLC(Prominence System; Shimadzu) on a reverse-phase column was equippedwith a PDA detector scanning from 200 to 800 nm and massspectrometry-ion trap-time of flight (MS-IT-TOF; Shimadzu) detector. Theinstrument and analysis condition show as follows.

Instrument: Shimadzu LC/MS-IT-TOF

Column: Atlantis T3, 2.1 mm I.D.×100 mm, 3 μm (Waters), 40° C.

Eluent: (A) 0.05% (v/v) Trifluoroacetic acid

-   -   (B) acetonitrile

Gradient: Eluent B. 10% (0 min)-25% (7.5 min)-100% (12.5 min)-100% (20min)

Flow Rate: 0.25 ml/min

Detector A: PDA 280 nm

Detector B: Atmospheric pressure chemical ionization (APCI)-MS(Negative-ion Mode; interface voltage, −3.0 kV, interface temperature,450° C.; curved desolvation line (CDL) temperature, 200° C.; nebulizerN2 gas, 2.0 L/min; drying N2 pressure, 70 kPa; heat block temperature,200° C.)

In the preliminary trials of LC/MS analysis, the electrospray ionization(ESI) was used as ionized probe, but the target compounds were not ableto observe in mass spectrum. When exchanging the ionization probe forAPCI and setting the interface temperature at 450° C., the mass spectrumof target compounds could be found out.

The mass spectrum data shows five peaks (FIG. 7). The [M-H]− at m/z401.0494 and 689.1135 were considered to be trifluoroacetic acid adductsof m/z 287.0553 and 575.1196 respectively. From these spectra, theparent mass of this compound appeared to be [M-H]− at m/z 575.1196,which was estimated to be C30H24O12 (error=0.17 ppm) that is an A-typedimer of procyanidin. Given the fact that strict conditions (APCI probetemperature at 450° C.) were required to ionize the compound, itappeared that the isolate was composed of one or more polymers ofprocyanidin.

5. Identification of Proanthocyanidin (PAC) by Butanol-HCl Method(Porter Method)

PAC were characterized by a modified method of Porter et al. (Porter etal., Phytochem. 25, (1986) 223; Shoji et al., J. Agric. Food Chem. 54,(2006) 884)

Heating PAC under acidic condition, carbenium ion and catechin areproduced by disconnecting the interflavan-bond thereof. The former aremore oxidized to give a red anthocyanidin. The porter method based onthe principle of these color reaction is able to perceptively detect PACfrom samples in increasing absorbance at 540 nm.

(1) Quantitative Determination and Qualitative Analysis of PAC by PorterMethod

200 μl of the purified compound from blueberry leaves was mixed with 750μl of n-butanol/HCl (95:5) and 50 μl of 1% of NH4Fe(SO4)2.12H2Odissolved in 2M HCl. The mixture was vortexed and heated in an oven at105° C. for 40 min and cooled in flowing water. Optical densities of thetreated solution were recorded at 540 nm by spectrophotometer (UV-1700,Shimadzu). Procyanidin B2 (Sigma-Aldrich) was used as a standard.

(2) LC/MS Analysis of Porter Method Products

The hydrolysates generated by the modified Porter method were alsoanalyzed using LC/MS-IT-TOF as follows.

Instrument: Shimadzu LC/MS-IT-TOF

Column: Atlantis T3, 2.1 mm I.D.×100 mm, 3 μm (Waters), 40

Eluent: (A) 0.5% (v/v) Formic acid containing 5 mM Ammonium Formate

-   -   (B) acetonitrile

Gradient: Eluent B. 10% (0 min)-40% (15 min)-100% (15 min)-100% (22.5min)

Flow Rate: 0.25 ml/min

Detector A: PDA 540 nm

Detector B: ESI-MS (Positive-ion Mode; interface voltage, 4.5 kV,interface temperature, 200° C.; curved desolvation line (CDL)temperature, 200° C.; nebulizer N2 gas, 1.5 L/min; drying N2 pressure,200 kPa; heat block temperature, 200° C.)

The purified LC3 fraction was analyzed by Porter method. The reactedsolution turned a red color, which corresponds to the color ofanthocyanidin generated by heating of procyanidin/proanthocyanidinmixture under acidic conditions. Using procyanidin B2 as the standard,the procyanidin content in the LC3 fraction was 86.33%. The hydrolysissolution was analyzed by LC/MS-IT-TOF. The main peak (RT=7.3 min) of thePDA chromatogram at 540 nm was observed at the same position as that ofthe cyanidin standard (FIG. 8A). Indeed, MS/MS spectra of this peak wereidentical to those of the cyanidin standard (FIG. 8B). These resultsrevealed that the HCV replication inhibitory compound in the LC3fraction from blueberry leaves was procyanidin and its derivatives. Asthe hydrolysate of this compound also contained a trace amount ofdelphinidin, these compounds was considered to be proanthocyanidinrather than procyanidin.

6. Analysis of Mean Degree of Polymerization (mDP) and Composition Unitsfor PAC by Thiolysis

Thiolysis was performed by previously described method with somemodifications (Guyot et al., J. Agric. Food Chem. 49, (2001) 14; Gu etal., J. Agric. Food Chem. 50, (2002) 4852). Briefly, 50 μl of PACsamples (2 mg/ml in methanol) was mixed with 50 μl of methanol acidifiedwith HCl (3.3%) and 100 μl of benzylmercaptan (5% in methanol). Thereaction was carried out at 50° C. for 30 min and then kept at ambienttemperature for 3 hours. Pure catechin or epicatechin solution (1.25mg/ml in methanol, Funakoshi) was also thiolysed to obtain theepimerization rate by calculating the ratio of catechin and epicatechinin the terminal units. The reaction mixture was diluted 5-fold withmethanol and analyzed by reverse-phase HPLC as follows.

Column: Atlantis T3, 4.6 mm I.D.×150 mm, 3 μm (Waters), 40° C.

Eluent: (A) 0.05% (v/v) Trifluoroacetic acid

-   -   (B) Acetonitrile

Gradient: Eluent B. 15% (0 min)-25% (10 min)-100% (40 min)-100% (45 min)

Flow Rate: 0.7 ml/min

Detector: 280 nm

To ascertain the elution pattern of thiolysis media and to estimateunknown peaks, LC/MS-IT-TOF was also employed in a negative ion mode.The instrument and analysis condition show as follows.

Instrument: Shimadzu LC/MS-IT-TOF

Column: Atlantis T3, 2.1 mm I.D.×100 mm, 3 μm (Waters), 40° C.

Eluent: (A) 0.05% (v/v) Trifluoroacetic acid

-   -   (B) acetonitrile

Gradient: Eluent B. 15% (0 min)-25% (10 min)-60% (40 min)-100% (40 min)

Flow Rate: 0.25 ml/min

Detector A: PDA 280 nm

Detector B: ESI-MS (Negative-ion Mode; interface voltage, −3.0 kV,interface temperature, 200° C.; curved desolvation line (CDL)temperature, 200° C.; nebulizer N2 gas, 1.5 L/min; drying N2 pressure,200 kPa; heat block temperature, 200° C.)

Flavan-3-ols and their benzylthio adducts obtained by thiolysate ofprocyanidin B2 was used as a standard. FIG. 9 shows the basic reactionof thiolytic cleavage. In thiolytic reaction, the PAC was reacted withbenzylmercaptan under the acidic condition so that the extension unitsof PAC were released to the corresponding benzylthioether and theterminal units were released to free flavan-3-ols.

As a result that thiolysis products of purified PAC in the LC3 fractionwere analyzed in reverse-phased HPLC, several peaks were identified(FIG. 10). The peaks A, C and H were considered to be catechin,epicatechin and benzylmercaptan respectively according to the retentiontime of each standard preparation. Other peaks were confirmed byanalyzing mass spectra.

The parent mass of peak E was [M-H]− at m/z 411.0892, with an estimatedformula C22H20O6S (error=−3.8 ppm), and its MS/MS spectrum was [M-H]− atm/z 287.0510. The difference between the parental mass and MS/MS was124.0382, which corresponded to a benzylthio adduct. Thus, peak Eappeared to be catechin or epicatechin benzylthioether. Furthermore,when procyanidin B2 which is dimer consisting of only epicatechin wasthiolysed, epicatechin and epicatechin benzylthioether as thiolysatewere detected from the reaction mixture. Since the retention time ofepicatechin benzylthioether from procyanidin B2 was the same as that ofpeak E, it was considered that that peak E was epicatechinbenzylthioether.

The parental mass of peak G was [M-H]− at m/z 697.1385 (predictedformula: C37H30O12S) and its MS/MS was [M-H]− at m/z 573.0987. Again,the difference therebetween was 124.0398 and likely represented thebenzylthio adduct. Thus, peak G was estimated to be a benzylthioether ofA-type dimer consisting of catechin and/or epicatechin.

According to Thompson et al. (Thompson et al., J. Chem. Soc. PerkinTrans. 1 (1972) 1387), the interflavan linkages in PAC were resistant tothiolysis. After thiolysis, they would be released as free A-typedimmers, if present in the terminal units or as the correspondingbenzylthioether, if present in the extension units (Foo et al., J. Nat.Prod. 63 (2000) 1225).

Peak B was detected as parent MS [M-H]− at m/z 863.1822 with a predictedformula C45H36O18 (error=−0.86 ppm). As the formula of B-typeprocyanidin trimer is C45H38O18 and that of A-type is C45H34O18, thispeak was likely a trimer in which A-type and B-type interflavan bondsco-existed.

Peak D was detected as parent MS [M-H]− at m/z 985.2009 and a predictedformula was removed from benzylthio adduct to C45H36O18. Peak D wassuggested to be an A-B type trimer similar to peak B but with abenzylthio adduct.

The parental mass of peak F was [M-H]− at m/z 605.1449 and its MS/MS was[M-H]− at m/z 481.1109, so that a benzylthio adduct was also present inpeak F. However, the predicted formula could not be obtained from theparental mass. Since the predicted formula of peak F was undefined, peakF is indicated as “unknown” in TABLE 3.

The thiolysates of PAC from blueberry leaves were obtained free AB-typetrimer, benzylthio adducts of AB-type trimer and A-type dimer other thanflavan-3-ol monomer and flavan-3-ol benzylthioether. Since theconstitution ratio of flavan-3-ol oligomer and their benzylthio adductswere high relatively, a calculating formula of mDP was modified.

The mean degree of polymerization (mDP) was calculated by the formula,mDP=[sum of (benzylthio adducts×n)+sum of (free flavan-3-ol×n)]/[totalfree flavan-3-ol], which “n” is DP of detected flavan-3-ol by thiolysis.Moreover, peak area of detected flavan-3-ol benzylthio adducts werecorrected from thiolysates of procyanidin B2 preparation.

The structural analysis of HCV inhibitor PAC from blueberry leaves (LC3)is summarized in TABLE 3. The mDP of PAC in this fraction was estimatedto be 7.7. The PAC from LC3 fraction was composed of primarilyepicatechin, which was 65.1% and 58.1% in terminal units and extensionrespectively. In addition, the monomer composition of the A-type dimerand the AB-type trimer were unknown. Therefore, when flavan-3-ol monomerproportion was re-calculated only in detected monomers, 95% of the PACfrom LC3 was composed of epicatechin.

TABLE 3 Thiolysis Results of Purified Fraction (LC3) from BlueberryLeaves Terminal (%) Extension (%) mDP C EC AB-3 Total C EC A-2 UnknownAB-3 Total LC3 7.7 20.4 65.1 14.5 100 0.8 58.1 11.9 23.2 6.0 100Notes: c; catechin, EC; epicatechin, AB-3; trimer consisting of bothA-type and B-type interflavan bonds, A-2; A-type dimer.

Example 2 Extraction of PAC Contained in Some of Material andReplication Suppressive Activities

1. PAC Preparation from Various Materials

From results of example 1, the proanthocyanidin (PAC) was identified ascompound possessing the suppression activity of HCV replication. PAC ispreviously known as condensed tannin and contained in various plants andfoods. It has been already reported that the following materials containPAC, so, PAC fraction can be separated from these materials.

Sample 1; blueberry leaves

Sample 2; blueberry fruit

Sample 3; taro peel

Sample 4; pine bark extract (Pycnogenol™)

Sample 5; grape seed extract (Gravinol™)

Sample 6; apple polyphenol (Applephenol™)

Sample 7; cranberry (cranberry powder)

Sample 8; strawberry fruit

Sample 9; peanut peel

Sample 10; croton sap (Sangre de Drago, Raintree Nutrition Inc.)

(Separation of PAC Fraction from Blueberry Leaves: Sample 1)

10 g of the lyophilized powder of rabbit-eye blueberry (Vacciniumvirgatum Aiton) leaves was extracted with 100 ml of hexane for 30 minand the supernatant therefrom was decanted. This procedure was repeatedthree times, followed by washing in 100 ml of ethyl acetate for 30 minthree times. The remaining residues were extracted with 100 ml ofmethanol for 30 min, and its supernatant was decanted and filtered. Thisprocedure was repeated four times and the resulting crude methanolextracts were concentrated by rotary evaporator at 50° C. andlyophilized, finally resulting in approximately 3.5 g of solid powder.

The crude methanol extract (approx. 500 mg) was then dissolved in 60%methanol and placed on a Sephadex LH-20 column (50 mm×50 mm; GEHealthcare). In fractionation, the following series of solvents wasused: 400 ml of 60% methanol; 400 ml of 100% methanol; 400 ml of 70%acetone. Each eluent were concentrated by evaporator and lyophilized,finally resulting in approximately 100 mg of solid PAC.

(Preparation of PAC Fraction from Sample 2˜10)

Sample 2, 3 and 9 was prepared directly from raw material and Sample 4to 8 and 10 was prepared from purchased materials containing PAC. Allmaterials were extracted with 100% methanol, and concentrated byevaporator and lyophilized. Then each PAC preparations were processed bythe same method aforementioned using Sephadex LH-20.

2. Composition Analysis of PAC Preparations

PAC samples prepared from each origin were analyzed by Porter method andthiolysis. Replicon cells were assayed for replication suppressiveactivity. These results are summarized as TABLE 4-6.

Table 6 shows the HCV replication suppressive activity, PAC contents andthe compositions of propelargonidin (PP), procyanidin (PC) andprodelphinidin (PD) which were calculated from results of thiolysis. Inthe samples showing no PAC contents value of table 6, there weredetected little quantity of the yielding PAC.

The mDP value of PAC preparations from blueberries (Sample 1 or 2) wasaround 12 and their composition had epicatechin-rich structure andA-type of interflavan bond. The mDP of PAC from taro peel (Sample 3) was10.5 and the most of them were epicatechin and possessed A-type bond. Inpycnogenol, mDP was approximately 6.5, and epicatechin was high incomposition ratio of extension units, but it was high catechin in thatof terminal units.

The gravinol (Sample 5) was characterized by that the mDP value hadrelatively-long about 14.4, and the composition ratio ofepicatechin-gallate was high in comparison with others. The mDP value ofthe applephenon (Sample 6) was approx. 4.4, same long as that ofstrawberry (Sample 8). Moreover, both samples (Sample 6 and 8) possessedhigh ratio of epicatechin composition, but epiafzelechin content wasapproximately 10% detected only in the strawberry (sample 8). The mDP ofthe cranberry fruit (Sample 7) and the peanut peel (Sample 9) were 6.6and 7.4 respectively, and both samples had comparatively high ratio ofA-type interflavan bond. The croton sap (Sample 10) was characterized inthat the mDP value was 8.3 and the composition ratio of epicatechin washigh, but gallocatechin and epigallocatechin were detected only in thePAC from the croton sap (sample 10), and the ratio became to 37.4% whengallocatechins were converted into prodelphinidin.

In HCV replication suppressive activities, the IC50 value of sample 1 to9 were less than 1.0 μg/ml, and the ratio was more than 10. It leads tothe conclusion in which these samples could be evaluated to have thesuppressive activity on HCV replication. But, in case of the croton sap(sample 10), the IC50 was more than 1.0 μg/ml, and the ratio of that wasless than 10, so that this was evaluated to have no activity.

From these results, almost all PAC from various plant origins wereidentified to have HCV replication suppressive activity in repliconcells, except PAC containing much prodelphinidin (PD) composition ratiossuch as croton sap (sample 10), because it could not be confirmed to hasthe suppressive activity.

TABLE 4 The mDP and Composition of PAC Preparations by ThiolysisTerminal Units Af EAf C EC GC EGC A-2 AB-3 mDP (%) (%) (%) (%) (%) (%)(%) (%) Total Blueberry Leaf 11.2 — — 31.2 42.6 — — — 26.2 100 BlueberryFruit 12.6 — — 43.6 56.4 — — — — 100 Taro Peel 10.5 — — — 100.0 — — — —100 Pycnogenol 6.5 — — 96.4 3.6 — — — — 100 Gravinol 14.4 — — 61.7 38.3— — — — 100 Applephenon 4.4 — — 10.7 89.3 — — — — 100 Cranberry Fruit6.6 — — — 41.8 — — 58.2 — 100 Strawberry 4.4 — 25.5 33.4 41.0 — — — —100 Fruit Peanuts Peel 7.4 — — 8.7 8.9 — — 77.0 5.4 100 Croton Sap 8.3 —— — 36.0 4.5 59.5 — — 100

TABLE 5 The mDP and Composition of PAC Preparations by ThiolysisExtension Units Af EAf C EC GC EGC Cg ECg Unknown A-2 AB-3 (%) (%) (%)(%) (%) (%) (%) (%) (%) (%) (%) Total Blueberry Leaf — — — 69.6 — — — —8.2 13.3 8.9 100 Blueberry Fruit — — — 85.6 — — — — 3.7 6.2 4.6 100 TaroPeel — — — 90.8 — — — — — 8.2 1.0 100 Pycnogenol — — 16.9 83.1 — — — — —— — 100 Gravinol — — 8.7 41.7 — — 2.8 46.8 — — — 100 Applephenon — — 3.594.1 — — —  2.4 — — — 100 Cranberry Fruit — — 1.4 68.5 — — — — — 19.510.5 100 Strawberry 0.7 4.9 19.0 75.4 — — — — — — — 100 Fruit PeanutsPeel — — 7.3 44.4 — — — — — 24.7 23.6 100 Croton Sap — 2.7 12.4 51.116.8 16.9 — — — — — 100

Af; afzelechin, EAf; epiafzelechin, C; catechin, EC; epicatechin, GC;gallocatechins, EGC; Epigallocatechin, Cg; catechin-gallate, ECg;epicatechin-gallate, A-2; A-type dimer, AB-3; trimer consisting of bothA-type and B-type interflavan bond.

TABLE 6 Anthocyanidin Components and Replicon Assay of PAC PreparationsComposition Replicon Assay PAC PP PC PD IC50 CC50 Content (%) (%) (%)(μg/ml) (μg/ml) Ratio (%) Blueberry Leaf — 100.0 — 0.56 16.26 29.0 88.1Bluebeny Fruit — 100.0 — 0.25 15.03 60.1 — Taro Peel — 100.0 — 0.1813.24 73.6 80.6 Pycnogenol — 100.0 — 0.56 18.88 33.7 65.0 Gravinol —100.0 — 0.20 9.95 49.8 55.9 Applephenon — 100.0 — 0.61 22.26 36.5 60.6Cranberry Fruit — 100.0 — 0.31 15.95 51.5 — Strawberry 10.2 89.8 — 0.2011.70 58.5 52.1 Fruit Peanuts Peel — 100.0 — 0.11 9.01 81.9 96.8 CrotonSap  2.4 60.2 37.4 5.89 23.62 4.0 64.9PP; propelargonidin, PC; procyanidin, PD; prodelphinidin

Example 3 The Bulk Preparation of PAC from Blueberry Leaves

To prepare PAC from blueberry leaves, freeze-dried powder (105 g) wasextracted with 1.2 liters of acetone for 10 min and the supernatantobtained was then decanted. This procedure was repeated five times toremove the green pigment from the leaves, followed by washing in 1.2liters of hexane for 10 min. The remaining residues were washed withethyl acetate. The washed powder of leaves was further extracted with1.2 liters of methanol for 30 min, and the supernatant obtained was thenfiltered. This procedure was repeated four times and the resulting crudemethanol extracts were concentrated by rotary evaporator at 50° C. andlyophilized, finally resulting in approximately 30 g of solid powder.

The crude methanol extract (15 g) was then dissolved in 1.0 liters of60% methanol and placed on a Sephadex LH-20 column (50 mm×920 mm; GEHealthcare). Fractionation was performed using the following series ofsolvents: fraction I, 9.0 L of 60% methanol (retrieved weight: 10.2 g);fraction II, 9.0 L of 100% methanol (retrieved weight: 3.3 g); fractionIII, 9.0 L of 70% acetone (retrieved weight: 1.3 g). In each fraction,the eluate was divided into 28 sub-fractions per liter.

After fractionation on a Sephadex LH-20 column, each eluate wasthiolysed to determine the components and mDP of PAC (FIG. 11). Then,PAC with different mDP from blueberry leaves was assessed for theinhibitory activity of HCV replication. The HCV replication suppressiveactivity of PAC from blueberry leaves was clearly dependent onpolymerization degree level and the apex activity was observed at apolymerization degree level of around 8 to 9. In this illustrativeembodiment, mDP of PAC was preferably at least 5.

(Discussion)

In FIG. 4 to 6, when the prodelphinidin units (R₁=R₂=R₃=OH) is high inPAC composition, the IC50 value of Sample 10 is more than 1.0 μg/ml andthe ratio is less than 10, so that it is not able to use as anti-HCVagent. In PAC composition of this invention, the degree ofpolymerization is more than or equal to 3, and delphinidin content waslow, so that HCV replication inhibition activity became strong, namelyit is useful as anti-HCV agent.

INDUSTRIAL APPLICABILITY

The HCV production suppressant of this invention possesses the superioranti-HCV effect, so that it is applied to therapeutic medicine ordietary supplements of hepatic disorders from HCV, instead of theinterferon therapy.

1. An agent for inhibiting production of hepatitis C virus comprisingproanthocyanidin polymer composition as an active ingredient, saidproanthocyanidin polymer composition having at least three monomerunits, each of which consists of the flavan-3-ol illustrated in thegeneral formula (1),

wherein R₁ is hydrogen or hydroxyl, R₂ is hydroxyl, R₃ is hydrogen whenR₁ is either hydrogen or hydroxyl, but R₃ is possibly hydroxyl when R₁is either hydrogen or hydroxyl to the extent that both R₁ and R₃ beinghydroxyl is at most 40 percents in the proanthocyanidin polymercomposition; said units of flavan-3-ol being bonded each other in anyone of three bond patterns as follows; (i) the bond between carbon atthe position 4 and carbon at the position 8, (ii) the bond betweencarbon at the position 4 and carbon at the position 6, (iii) the bondbetween carbon at the position 4 and carbon at the position 8, andbetween carbon at the position 2 and oxygen at the position
 7. 2. Anagent according to claim 1, wherein the R₄ of the monomer unit offlavan-3-ol is a gallate group.
 3. An agent according to claim 1,wherein the ratio between the concentration of proanthocyanidin polymercomposition inhibiting 50 percents of hepatitis C virus proliferationactivity and the concentration of proanthocyanidin polymer compositioninhibiting 50 percents of cell proliferation activity is at most onetenth.
 4. An agent according to claim 1, wherein the proanthocyanidinpolymer composition is 5 to 10 monomer units of flavan-3-ol.
 5. An agentaccording to claim 4, wherein the 5 to 10 monomer units of flavan-3-olare bonded each other in any one of the three bonding patterns (i), (ii)and (iii).
 6. An agent according to claim 1, wherein theproanthocyanidin polymer composition comes from blueberry leaves.
 7. Anagent for treating hepatic disease arisen from hepatitis C viruscomprising the agent for inhibiting production of hepatitis C virusaccording to claim
 1. 8. An agent for inhibiting onset or development ofhepatic disease arisen from hepatitis C virus comprising the agent forinhibiting production of hepatitis C virus according to claim
 1. 9. Amedical composition or a prodrug for inhibition of onset or development,or treatment of hepatic disease arisen from hepatitis C virus comprisingthe agent for inhibiting production of hepatitis C virus according toclaim
 1. 10. A dietary supplement for inhibiting onset or development ofhepatic disease arisen from hepatitis C virus comprising the agent forinhibiting production of hepatitis C virus according to claim 1.